Experimental genomics in combination with the growing body of sequence information promise to revolutionize the way cells and cellular processes are studied. Information on genomic sequence can be used experimentally with high-density DNA arrays that allow complex mixtures of RNA and DNA to be interrogated in a parallel and quantitative fashion. DNA arrays can be used for many different purposes, most prominently to measure levels of gene expression (messenger RNA abundance) for tens of thousands of genes simultaneously. Measurements of gene expression and other applications of arrays embody much of what is implied by the term ‘genomics’; they are broad in scope, large in scale, and take advantage of all available sequence information for experimental design and data interpretation in pursuit of biological understanding.

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Genomics, gene expression and DNA arrays.

https://www.cell.com/molecular-cell/pdf/S1097-2765(00)00051-4.pdf

A Regulatory Cascade of the Nuclear Receptors FXR, SHP-1, and LRH-1 Represses Bile Acid Biosynthesis

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays

The catabolism of cholesterol to bile acids represents a major pathway for the elimination of this potentially pathogenic sterol fromthe body. Bile acids subserve a number of important physiological functions, including the solubilization of cholesterol, fat soluble vitamins, and other lipids in the intestine (Vlahcevic et al. 1994, 1996). In addition, bile acids contribute to the generation of bile flow and promote the secretion of lipids, notably phosphatidylcholine and cholesterol, fromthe canalicular membrane into the bile canaliculus. However, because of their intrinsic toxicity, intracellular levels of bile acids must be tightly regulated, which is largely accomplished by transcriptional regulation of genes encoding proteins involved in bile acid biosynthesis, transport, and metabolism.

The conversion of cholesterol to the primary bile acids, cholic acid and chenodeoxycholic acid (CDCA), involves at least 14 distinct enzymes and is accomplished via 2 pathways (Bjorkhem 1985; Russell and Setchell 1992). The first and rate-limiting step in the neutral (classic) pathway of bile acid biosynthesis is catalyzed by cholesterol 7 α-hydroxylase (CYP7A1; Bjorkhem 1985; Russell and Setchell 1992; Chiang 1998). Expression of the gene encoding CYP7A1 is known to be suppressed by a number of factors including insulin, protein kinase C activators, cytokines such as tumor necrosis factor α (TNF-α), steroid hormones, and, importantly, bile acids (for review, see Chiang 1998). The bile acid-dependent feedback repression of CYP7A1 is important in preventing a potentially harmful expansion of the bile acid pool. A number of studies have focused on characterizing the molecular mechanisms by which bile acids suppress CYP7A1 expression, and it is now apparent that multiple, redundant pathways exist (Stravitz et al. 1995; Antes et al. 2000; Goodwin et al. 2000; Lu et al. 2000; Miyake et al. 2000; De Fabiani et al. 2001; Kerr et al. 2002; Wang et al. 2002). Notably, these signaling cascades converge on a common bile acid responsive element (BARE) in the CYP7A1 promoter (Chiang and Stroup 1994; Stroup et al. 1997). This element is highly conserved across species and is a well-documented binding site for members of the nuclear receptor superfamily of ligand activated transcription factors, including liver receptor homolog-1 (LRH-1, NR5A2) and hepatocyte nuclear factor 4α (HNF-4α, NR2A1; Crestani et al. 1998; Nitta et al. 1999; Lu et al. 2000; Stroup and Chiang 2000; De Fabiani et al. 2001; Chiang 2002).

The farnesoid X receptor (FXR; NR1H4) is a bile acid-activated transcription factor that also belongs to the nuclear receptor family (Makishima et al. 1999; Parks et al. 1999; Wang et al. 1999). FXR binds DNA as an obligate heterodimer with the retinoid X receptors (RXRs; Forman et al. 1995; Seol et al. 1995). The FXR/RXR heterodimer typically binds to an inverted repeat of the hexanucleotide motif AGG/TTCA separated by a single nucleotide, a so-called IR-1 (Forman et al. 1995; Seol et al. 1995). FXR is known to be expressed in tissues that are exposed to bile acids, including liver, intestine, gallbladder (C. Housset, pers. comm.), kidney, and adrenal gland (Forman et al. 1995; Seol et al. 1995). In liver, the biological consequences of FXR activation have recently become increasingly clear. Upon activation, FXR initiates transcription of a cohort of genes that function to decrease the concentration of bile acids within the hepatocyte. Specifically, FXR induces the expression of ATP-binding cassette (ABC) transporters bile salt export pump (BSEP; ABCB11; Sinal et al. 2000; Ananthanarayanan et al. 2001; Plass et al. 2002), multidrug resistance protein 3 (MDR3, ABCB4; B. Goodwin and S.A. Jones, unpubl.), and multidrug resistance-associated protein 2 (MRP2; ABCC2; Kast et al. 2002). These transporters function to transport bile acids and bile constituents fromthe hepatocytes into the bile. In addition, activation of FXR by both naturally occurring (CDCA) and synthetic ligands leads to the repression of two important genes in the bile acid biosynthetic pathway, namely CYP7A1 and CYP8B1, which encodes oxysterol 12α hydroxylase (Goodwin et al. 2000; Lu et al. 2000; Sinal et al. 2000; del Castillo-Olivares and Gil 2001; Zhang and Chiang 2001). The FXR-dependent suppression of CYP7A1 is mediated by the transcriptional repressor short heterodimer partner-1 (SHP; NR0B2), an atypical nuclear receptor that lacks a DNA-binding domain (Goodwin et al. 2000; Lu et al. 2000). Thus, activation of FXR results in increased expression of the SHP gene. In turn, SHP interacts with LRH-1, a known positive regulator of CYP7A1 (discussed above) and represses its transcriptional activity. Elegant studies performed in mice harboring a disrupted SHP gene confirmthe importance of the FXR–SHP–LRH-1 cascade in suppression of CYP7A1, however, they also demonstrate the existence of additional SHP-independent pathways, possibly involving the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (Kerr et al. 2002; Wang et al. 2002).

In this study, we describe the discovery of a novel FXR-dependent signaling cascade for the suppression of CYP7A1. We show that FXR directly regulates expression of FGF-19, a member of the fibroblast growth factor (FGF) family of signaling molecules (Nishimura et al. 1999; Xie et al. 1999). The FGFs bind the extracellular domain of their cognate cell surface receptor (FGFR) and induce receptor dimerization and tyrosine kinase phosphorylation, which, in turn, leads to the activation of a number of intracellular pathways (Goldfarb 2001; Ornitz and Itoh 2001). For many years, it has been understood that the FGFs regulate cell growth, differentiation, and morphogenesis, however, it is now apparent that some of these proteins are also important components of specific homeostatic pathways (Yu et al. 2000; Shimada et al. 2001; Tomlinson et al. 2002). We demonstrate that FGF-19, acting as an FXR-induced signaling molecule, represses expression of the CYP7A1 gene. Our findings define a novel regulatory pathway for the suppression of bile acid biosynthesis.

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Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis

Average maize yields have increased steadily over the years in the USA and yet the variations in harvestable yield have also markedly increased. Much of the increase in yield variability can be attributed to (1) varying environmental stress conditions; (2) improved nitrogen inputs and better weed control; and (3) continuing sensitivity of different maize lines to the variation in input supply, especially rainfall. Drought stress alone can account for a significant percentage of average yield losses. Yet despite variable environments, new commercially available maize hybrids continue to be produced each year with ever-increasing harvestable yield. Since many factors contribute to high plant performance under water deficits, efforts are being made to elucidate the nature of water-stress tolerance in an attempt to improve maize hybrids further. Such factors include better partitioning of biomass to the developing ear resulting in faster spikelet growth and improved reproductive success. An emphasis on faster spikelet growth rate may result in a reduction in the number of spikelets formed on the ear that facilitates overall seed set by reducing water and carbon constraints per spikelet. To understand the molecular mechanisms for drought tolerance in improved maize lines better, a variety of genomic tools are being used. Newer molecular markers and comprehensive gene expression profiling methods provide opportunities to direct the continued breeding of genotypes that provide stable grain yield under widely varied environmental conditions.

Molecular and physiological approaches to maize improvement for drought tolerance

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

Gene expression analysis by transcript profiling coupled to a gene database query.

Disclosed are methods for identifying nucleic acids in a sample of nucleic acids in which nucleic acids are initially present in unequal amounts. The methods include partitioning the starting population of nucleic acids to form one or more subpopulations, and then identifying nucleic acids that are present in different amounts in the partitioned nucleic acid sample as compared to the starting population.

Method of identifying nucleic acids

Disclosed are methods for identifying nucleic acids in a sample of nucleic acids in which nucleic acids are initially unequal amounts. The methods include partitioning the starting population of nucleic acids to form one or more subpopulations, and then identifying nucleic acids that are present in different amounts in the partitioned nucleic acid sample as compared to the starting population. Also disclosed are methods of generating databases based on sizes of DNA fragments and size-based sequencing of those fragments.

Paul Predki

Papers

Gene expression analysis by transcript profiling coupled to a gene database query.

Method of identifying nucleic acids

Method for identifying a biomolecule